Cephalosporin Substrate Specificity Determinants of TEM-1 β-Lactamase

Abstract

␤-lactamase is a bacterial enzyme that catalyzes the hydrolysis of ␤-lactam antibiotics such as penicillins and cephalosporins. TEM-1 ␤-lactamase is a prevalent ␤-lactamase found in Gram-negative bacteria and is capable of hydrolyzing both penicillins and cephalosporins, except for the extended-spectrum cephalosporins. To identify the sequence determinants in the active site for a given antibiotic substrate, random libraries were constructed that each contain all possible amino acid combinations for the designated region of TEM-1 ␤-lactamase. To establish the determinants of substrate specificity for cephalosporins versus those for penicillins, these active site libraries have been screened for mutants with high levels of activity for the second generation cephalosporin cephaloridine. Based on the sequence results, substitutions of W165S, A237T, and E240C were identified as cephalosporin-specific. Kinetic analysis of these mutants was done to determine whether each is capable of distinguishing between the two classes of antibiotics. Both the A237T and E240C substitutions, alone or in combination, exhibited increased cephalosporinase activity and decreased penicillinase activity relative to the wild-type enzyme. A sequence comparison between functional mutants selected for cephaloridine hydrolytic activity and functional mutants previously selected for ampicillin hydrolytic activity suggests that TEM-1 ␤-lactamase has greater restrictions in maintaining cephalosporinase activity versus maintaining penicillinase activity.␤-lactam antibiotics, such as the penicillins and cephalosporins, are among the most commonly used antimicrobial agents. The production of ␤-lactamases, which catalyze ␤-lactam hydrolysis, is the predominant mechanism of bacterial resistance to these antibiotics (1). ␤-lactamases are grouped into 4 classes (A, B, C, and D) based on primary sequence homology (2, 3). Classes A, C, and D are serine hydrolases (3) while class B consists of zinc metalloenzymes (4). TEM-1 ␤-lactamase is a plasmid encoded, class A enzyme that can efficiently cleave the penicillins and some cephalosporins (5). The third generation or extended-spectrum cephalosporins, such as ceftazidime and cefotaxime, however, are poor substrates for the TEM-1 enzyme (6).Changes in the substrate specificity of TEM-1 ␤-lactamase due to amino acid substitutions in the enzyme have been observed among resistant clinical isolates in response to selective pressure applied by extended-spectrum antibiotics (6). The most common amino acid substitutions occur at residues 104, 164, and 237-240, both as individual and as combinatorial substitutions (numbering of amino acids according to Ambler et al. (7)). The substitutions are thought to provide more favorable interactions between the enzyme and the bulkier side chains of the extended-spectrum cephalosporins, thus increasing the catalytic efficiency of the enzyme (6).Because of the importance of mutation-meditated changes in TEM-1 ␤-lactamase substrate specificity during the development of antibiot...

Acyl-intermediate Structures of the Extended-spectrum Class A β-Lactamase, Toho-1, in Complex with Cefotaxime, Cephalothin, and Benzylpenicillin

“…5, A and B). However, the fact that a W165S mutant of TEM-1 does not hydrolyze cefotaxime (35) indicates the likelihood of multiple factors influencing the unfavorable disposition of the ⍀-loop in non-ESBLs.…”

Amino Acid Substitutions at Ambler Position Gly238 in the SHV-1 β-Lactamase: Exploring Sequence Requirements for Resistance to Penicillins and Cephalosporins

“…The difference we observed in MICs between cephalosporins and penicillins raises the question: is the ABL238 position "cephalosporin specific" in SHV (i.e., greater cephalosporinase activity versus penicillinase activity)? In TEM, Ala237Thr and Glu240Cys assume this role (4,5).…”

E240V Substitution Increases Catalytic Efficiency toward Ceftazidime in a New Natural TEM-Type Extended-Spectrum β-Lactamase, TEM-149, from Enterobacter aerogenes and Serratia marcescens Clinical Isolates

“…In the study described in this paper, we correlated the kinetic parameters of the TEM-149 and the TEM-149 T182M enzymes with those reported by Raquet et al for the well-characterized TEM-10 enzyme (19). This is the first finding of a valine at position 240, which has never been described in other natural variants or laboratory mutants belonging to the TEM, SHV, and CTX-M families (4,5,26). In this study we also report on the purification and kinetic analysis of TEM-149 T182M , a TEM-149 mutant with a reversion of a residue at position 182.…”